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| Author(s): |
Isadora Carolina Betim Pavan
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Limeira, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Aplicadas |
| Defense date: | 2017-08-30 |
| Examining board members: |
Fernando Moreira Simabuco;
Sandra Martha Gomes Dias;
Patrícia de Oliveira Prada
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| Advisor: | Fernando Moreira Simabuco |
| Abstract | |
Introduction: The mTOR signaling pathway has been related to several diseases and metabolic disorders in humans, including obesity, diabetes, several types of cancer and neurodegeneration. The S6Ks proteins have shown an important role in mTOR signaling, acting as effectors of this pathway. The S6Ks family is composed of two genes, RPS6KB1 and RPS6KB2. RPS6KB1 encodes two isoforms: p85- and p70-S6K1, while RPS6KB2 encodes two other isoforms, known as p56 and p54-S6K2. This study investigated the interaction partners of p70-S6K1 and p54-S6K2 isoforms in HEK293 cells, in order to differentiate these proteins according to their involvement in biological processes. Furthermore, it was investigated in more detail the interaction of p70-S6K1 and p54-S6K2 isoforms with eIF2? and PARP1 proteins. Justification: It was believed that S6K1 and S6K2 exhibited redundant biological functions, since they have a high degree of similarity. Thus, few studies aim to identify different roles and implications of these isoforms in biological processes. However, discovering different functions of these proteins may have great importance for the investigation of new strategies to develop therapies for certain types of diseases such as cancer. In general, this work contributes to a better understanding of the mTOR / S6Ks pathway. Results: The first data obtained from this study, which generated a scientific article published in the journal Proteomics in 2016 (Annex I), it was observed, by mass spectrometry, that the p70-S6K1 and p54-S6K2 isoforms have different interaction proteins and act in different biological processes, indicating that such isoforms have distinct biological functions. Some partner interaction, as eIF2?, PARP1, PPM1B, FXR1, FMRP, PRMT5 and WDR77, were chosen for its relations with the p70-S6K1 and p54-S6K2 isoforms were studied in more detail. Some immunoprecipitation experiments were performed showing evidence of the interaction between p70-S6K1 and p54-S6K2 with the eIF2? protein, and only p54-S6K2 with the PARP1 protein. In a situation of cellular stress, eIF2? is phosphorylated at its serine residue 52 (S52), leading to the inhibition of global protein synthesis and the stimulation of autophagy and apoptosis. We have identified that eIF2? has a predicted phosphorylation site of S6Ks (RXRXXT/S) at serine 58 (S58), which is conserved in a variety of species. Immunoprecipitation assays revealed that eIF2? is phosphorylated at RXXT/S site in the presence of insulin. In addition, it was shown that the activation of S6K1 and S6K2 through insulin and only S6K2 through FGF2 is directly related to the reduction in eIF2? phosphorylation in serine 52. Conversely, pharmacological inhibitors of mTOR and S6Ks, such as rapamycin and PF4708671, respectively, are associated with increased of eIF2? phosphorylation levels at S52. The overexpression of constitutively active p70-S6K1 and p54-S6K2 was also performed and reduction of the phosphorylation of eIF2? (S52) was seen. Finally, a site-directed mutation assay was performed on eIF2?, mimicking a phosphorylated residue 58 of eIF2?. As a result, we observed that constant phosphorylation at residue 58 is accompanied by a reduction of phosphorylation of residue S52. Conclusions: This study demonstrated that S6Ks isoforms present different interacting proteins, indicating that those isofroms present distinct biological functions in the cell. The results also suggest that S6Ks can phosphorylate eIF2? in serine 58, and due to the proximity of the sites may be an inhibition of the phosphorylation of serine 52, thereby regulating the activity eIF2á (AU) | |
| FAPESP's process: | 15/00311-1 - Functional characterization of interacting proteins of different S6Ks isoforms |
| Grantee: | Isadora Carolina Betim Pavan |
| Support Opportunities: | Scholarships in Brazil - Master |